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<div><em>Agrobacterium tumefaciens</em> causes crown gall disease on plants via transforming T-DNA into the plant genome and is a useful tool for genetic modification. To clarify novel plant components which regulate the transformation process, we used <em>Arabidopsis thaliana </em>seedlings as a model system to reveal the gene expression profiles on <em>Agrobacterium</em> infection. Our transcriptome analysis of <em>Agrobacterium</em>-infected seedlings identified the differential regulation of genes involved in metabolism of secondary metabolites glucosinolates (GSs) and camalexin, suggesting their potential roles in regulating <em>Agrobacterium</em> transformation processes. Thus, we evaluated the impacts of GSs and camalexin during different stages of <em>Agrobacterium-</em>mediated transformation combining <em>Arabidopsis </em>mutant studies, metabolite profiling, and exogenous applications of various GS hydrolysis products or camalexin. The results showed that indole GS hydrolysis played an inhibitory role in <em>Agrobacterium-</em>mediated transient transformation efficiency on <em>Arabidopsis </em>seedlings. Later in the <em>Agrobacterium</em> infection process, camalexin accumulation was a key factor inhibiting tumor development on <em>Arabidopsis </em>inflorescence stalks. No correlation could be found between transformation efficiency and bacterial growth and/or colonization upon GSs and camalexin applications, suggesting their impacts on plant transformation likely function as signals in regulating plant defense. Based on our findings, we suggest the hydrolysis products of indole GSs are used by <em>Arabidopsis</em> to defend <em>Agrobacterium</em> at early stage, and camalexin inhibits tumor development at the later stages. In addition, the differential effects of certain GS hydrolysis metabolites on <em>Agrobacterium</em> transformation may be used to control crown gall diseases or to enhance the transformation efficiency.</div>